Within the tryptophan operon (7000 base pairs of DNA) of Escherichia coli are found at least a dozen regions where simple mutational changes can generate sites for the unregulated initiation of transcription (promoters). Such promoters are readily recognized through changes in phenotype: first there are elevated levels of downstream enzyme which can be specifically attributed to the presence of a promoter not found in wild-type trp DNA; second, new promoter mutations frequently inactivate a trp structural gene, thus making it possible to score a promoter by observing tryptophan auxotrophy. After isolating short fragments of DNA containing new promoters, the base sequence will be determined using the method of Maxam and Gilbert. By comparing the sequence so ascertained with other constitutive promoters, we expect to gain new insight into an important facet of gene expression. The nature of those cytoplasmic elements which function in promoter recognition will be defined by identifying other genetic loci which can modify promoter activity. We have shown that mutational modifications of the beta subunit of RNA polymerase (selected by taking advantage of the rifamycin-resistant nature of rpoB mutations) can alter the recognition of internal trp promoters and have thus established that all elements of the promoter-polymerase interaction system can potentially be mapped. Structure-function relationships within the trpR gene-Trp repressor system will be elucidated through the isolation and characterization of trpR DNA and with the aid of a selective system which enables us to generate unlimited numbers of deletion mutants with endpoints throughout the trpR gene.